The ability of copper (Cu) to act as an electron transfer intermediate makes it an important co-factor in a variety of enzymes that perform essential biochemical functions; however, this same' property is also responsible for its potent cytotoxicity. Thus, it is critical that organisms maintain a tightly regulated Cu sensing mechanism to maintain the delicate balance between essential and toxic levels of Cu. The studies outlined in this proposal are aimed at characterizing a new member of the Cu Metalloregulatory Transcription Factor (CuMRTF) family, designated MRTF-X. The known members of the yeast CuMRTF family, ACE1, AMT, and MAC1 directly sense Cu and respond by activating the transcription of metal detoxification genes known as metallothioneins at high Cu concentrations, and a Cu,Fe reducing gene known as FRE1 which is required for high affinity Cu transport at low Cu concentrations. The partial sequence of the amino terminal 110 amino acids of MRTF-X is 44% and 53% identical and 65% and 68% similar to the same region in AMT1 and ACE1, respectively which is involved in a Cu-dependent DNA binding activity. In this application, the structure, function, expression, subcellular location, and target DNA sequences of MRTF-X will be characterized by gene deletion mutagenesis, DNA binding assays, Northern blot analysis, in vivo gene expression assays and indirect immunofluorescence microscopy. This study will provide important information on the mechanisms involved in the transcriptional regulation of the genes that maintain normal Cu homeostasis.